Complex carbohydrates expressed by some pathogens facilitate the evasion of host immune responses and contribute to the destructive sequelae following infection with organisms such as Streptococcus pneumoniae, Neisseria meingititidis, Haemophilus influenza, Salmonella typhi, and human immunodeficiency virus (“HIV”). In particular, carbohydrate rich regions of glycoproteins are poorly immunogenic. Factors contributing to this lack of immunogenicity include dilution of single antigenic response due to carbohydrate microheterogeneity and steric interference with highly immunogenic protein epitopes. See, e.g., Rudd, et al., Crit. Rev. Biochem. Mol. Biol. 32:1-100 (1997); Woods, et al., Nature Struct. Biol. 1:499-501 (1994). Additionally, viruses frequently fool the host immune system by using the cellular glycosylation machinery to express endogenous glycans for which the host is already tolerized.
The role of glycoproteins in HIV pathogenicity, particularly that of gp120, exemplifies the difficulty and importance of complex carbohydrates in eliciting protective host immune responses. HIV, like all viruses, requires the transcriptional and translation machinery of a cell in order to successfully propagate itself in the host. HIV accomplishes this by entering a cell through interactions with specific cellular receptors. Specifically, the HIV virion enters a cell, usually a T lymphocyte, through the interaction of its viral envelope protein (“Env”) with the cellular CD4 receptor and the CCR5 cellular coreceptor during early infection, and through the CD4 receptor-CXCR4 coreceptor complex later in infection. See, e.g., Pöhlmann, et al., J. Virol. 75:4664-72 (2001); Teunis, et al., Cell 100:587-97 (2000). The Env protein is a glycoprotein known as gp120. On the surface of the HIV virion, three molecules of gp120 are noncovalently linked to another cell surface protein, gp41. See, e.g., Zolla-Pazner, Nature Rev. Immunol. 4:199-210 (2004). gp41 is a transmembrane glycoprotein found as a homotrimeric complex in the viral envelope. This gp120-gp41 complex forms hetero-oligomeric spikes on the HIV virion that first binds the CD4/coreceptor complex and then subsequently undergoes a conformational change resulting in the exposure of the viral fusion peptide that mediates the entry of the virus into the cell.
Recent evidence suggests that a second interaction distinct from the CD4/coreceptor interaction is also critical to HIV transmission and infection, particularly at the earliest stages. Dendritic cells (DCs), a highly specialized antigen presenting cell, is the first cell targeted by HIV upon infection. See Geijtenbeek, et al., Cell 100:587-97 (2000). DCs at the mucosa capture, internalize, and transport HIV from the mucosal surfaces to remote lymph nodes via an interaction between gp120 and DC-SIGN, a cell surface molecule on DCs. The delivery of intact virus by the DCs then results in the infection of CD4+ T-lymphocytes. See Bashirova, et al., J. Exp. Med. 193:671-78 (2001). In other words, DC-SIGN acts in trans to mediate efficient infection of CD4+ cells by HIV. See Pöhlmann, et al., J. Virol. 75:4664-72 (2001). Later studies showed that DC-SIGN also acts in cis to promote efficient viral infection. See, e.g., Lee, et al., J. Virol. 75:12028-38 (2001).
Given the difficulty in eliminating infected cells, an immune response is likely to be most effective if the elicited response impedes the entry of the virus into the cell. To date, attempts to elicit such protective responses have been hampered by the poor immunogenicity of gp120. gp120 contains extensive glycosylation and highly variable loops interspersed with more conserved, functionally constrained regions acting as physical shields for critical gp120 epitopes, i.e., those epitopes that interact with the receptor/coreceptor complex, from antibodies that can block viral entry or neutralize the virus. See, e.g., Garber, et al., Lancet Infect. Dis. 4:397-413 (2004). However, naturally elicited neutralizing antibodies have been identified, confirming the potential for effective neutralizing responses. See, e.g., Burton, et al., Nature Immunol. 5:233-36 (2004).
Development of HIV/AIDS vaccine to induce neutralizing antibodies against a broad spectrum of HIV-1 primary isolates is still a highly challenging endeavor, 25 years after the discovery of AIDS. The challenge for developing effective vaccines lies in the identification of appropriate antigenic epitopes that can be presented immunogenically such that neutralizing antibodies are elicited in the host. The challenge in developing successful HIV vaccines is further complicated by an expansive diversity in primary HIV isolates. See, e.g., Gaschen, et al., Science 296:2354-60 (2002). To date, traditional approaches to vaccine design have not proven successful in eliciting neutralizing antibody responses. Among the approximately 30 clinical trials of HIV vaccines, none are able to induce broadly neutralizing antibodies. One of the major challenges is the lack of an appropriate design of an antigen with neutralizing epitopes that are exposed on the surface of the antigen and highly conserved in most or all subtypes of HIV-1. Pantophlet R et al., Annu Rev Immunol. 24:739-69, 2006. Up to date only four monoclonal antibodies (MAbs) with broad and potent neutralizing activity were isolated from HIV-1 infected humans. Douek D C et al., Cell 124:677-81 (2006). None of them can be duplicated in all tested species of animals. Among the four MAbs, one targets a conformational epitope on the HIV-1 env gp120, two recognize gp41, and one, 2G12, binds to the high mannose-type carbohydrates on gp120.
The earliest target cell for HIV infection is the dendritic cell (DC), and therefore the most potent vaccine is one that disrupts the ability of HIV to target DCs in a host. The high mannose oligosaccharides of gp120 provide epitopes essential for HIV-DC interaction, and thus provide suitable vaccine targets. High mannose oligosaccharides mediate the interaction between DC-SIGN and gp120. See Geijtenbeek, et al., Cell 100:587-97 (2000). Yet, the naturally occurring gp120 only expresses about 20% Man8GlcNAc2 (Man8) and 10% Man9GlcNAc2 (Man9). See Scalan, et al., J. Virol. 76:7306-21 (2002). Cyanovirin-N(CV-N), a cyanobacterial protein, binds to high mannose oligosaccharides of gp120, specifically recognizing the Manα1,2-Man structures on Man9GlcNAc2 (Man9) and the D1 D3 isomer of Man8GlcNAc2 (Man8), and through this interaction acts as a potent microbicide against HIV. See Bewley, et al., J. Am. Chem. Soc. 123:3982-902 (2001); Sandstrom, et al., Biochem. 43:13926-31 (2004). Furthermore, one of the naturally occurring, neutralizing antibodies specifically recognizes a cluster of Manα1,2-Man high mannose oligosaccharides of gp120. See Scanlan, et al., J. Virol. 76:7306-21 (2002). This antibody, known as the 2G12 antibody, potently neutralizes a broad range of HIV-1 primary isolates by inhibiting the HIV virion interaction with DCs and CD4+ T cells. See, e.g., Trkola, et al., J. Virol. 70:1100-08 (1996); Sanders, et al., J. Virol. 76:7293-305 (2002).
High Mannose Type Glycans as a Target for HIV-1 Vaccine
Development of a carbohydrate-based HIV vaccine is considered to be one of the novel approaches for a prophylactic vaccine. Wang, Curr Opin Drug Discov Devel. 9(2):194-206, 2006. Several lines of evidence have demonstrated that the terminal Manα1,2-Man structures (α1,2-linked mannose) found on the D1 and D3 arm of Man8NAcGlc2 present novel targets on the gp120 glycoprotein, with the possibility of inducing potent, neutralizing antibodies against HIV-1 from different strains and subtypes.
High-mannose glycans on gp120 are recognized by the broadly neutralizing MAb 2G12. Among the hundreds of MAbs against gp120 that have been generated in rodents and isolated from HIV-1 infected humans, 2G12 is the only one that recognizes virus carbohydrates and potently neutralizes a broad range of HIV-1 primary isolates. It does so by inhibiting the interactions of HIV-1 with DCs and CD4+ T cells. Trkola et al., J. Virol. 70(2):1100-8, 1996; Scanlan et al., J. Virol. 76(14):7306-21, 2002; Sanders et al, J. Virol. 76(14):7293-305, 2002; The binding site of 2G12 has been identified as high mannose-type glycans on the HIV-1 env gp120 glycoprotein. More specifically, the 2G12 MAb binds to a cluster of terminal α1,2-linked mannose residues from at least three high-mannose glycans (Scanlan et al., J. Virol. 76(14):7306-21, 2002); it does not recognize other carbohydrates or mannose residues with different terminal linkages, α1,3-linked or α1,6-linked mannose.
The Manα1,2-Man structures on high-mannose glycans are also the binding sites for a potent HIV-1 inhibitor. The cyanobacterial protein termed Cyanovirin-N(CV-N) can inactivate a diverse array of laboratory strains and primary isolates of HIV-1, HIV-2 and SIV. Boyd et al., Antimicrob Agents Chemother. 41(7):1521-30, 1997. The protein can also block HIV-1 gp120 interaction with CD4 and coreceptors, prevent virus-to-cell fusion, and stop infection of cells. Esser et al., J. Virol. 73(5):4360-71, 1999. Dey et al., J. Virol. 74(10):4562-9 2000. These potent properties of CV-N are attributed to its ability to bind with extremely high affinity to the high-mannose oligosaccharides on gp120. Specifically, the inhibitor recognizes the Manα1,2-Man structures on Man9GlcNAc2 (Man9) and the D1 D3 isomer of Man8GlcNAc2 (Man8), but not other forms of high mannoses, including Man7, Man6, and Man5. Bewley et al., J Am Chem. Soc. 123(17):3892-902.2001; Sandstrom et al., Biochemistry. 43(44):13926-13931 2004. This ability of CV-N to inhibit HIV infection presents evidence of the potency of such molecules that are able to bind to these terminal glycan structures.
Dendritic cells have been shown to enhance infection through the interaction of DC-SIGN with the high-mannose glycans on gp120. Recently, DCs were found to be the first cell type targeted by HIV in the body. DCs at the mucosa are found to capture, internalize and transport HIV to remote lymph nodes where they deliver the intact virus to CD4+ T-lymphocytes. Geijtenbeek et al., Cell. 100:587-97, 2000. It was found that all tested strains of HIV-1, HIV-2, SIV and SHIV bind to DCs, with DC-SIGN playing an important role in this process. Pohlmann, et al., J. Virol. 75(10):4664-72, 2001. The interaction between HIV and DC-SIGN is mediated by the high-mannose glycans on gp120. Geijtenbeek et al., Cell. 100:587-97, 2000. In fact, synthetic high mannose oligosaccharides are able to bind DC-SIGN and prevent subsequent HIV interactions. Feinberg et al., Science. 294(5549):2163-6, 2001.
The gp120 protein of HIV-1 is heavily glycosylated and contains an average of 25 N-linked glycosylation sites. Approximately half of them are occupied by high mannose-type or hybrid-type glycans (Leonard et al., J Biol. Chem. 265(18): 10373-82, 1990), with the high mannose glycans interacting with 2G12, CV-N, and DC-SIGN through different binding sites. MAb 2G12 binds a cluster of D1 arms from at least three Man9 or Man8 residues, CV-N binds with a high affinity to the D1 and D3 arms from a single Man9 and Man8 residues, and DC-SIGN binds several mannoses residues through its tetramer.
Altogether, these results indicate the strong possibility of inhibiting an early stage of HIV infection with highly specific neutralization antibodies against the Manα1,2-Man structures found on high mannose glycans. The major challenge is to develop an antigen containing strictly high mannoses with terminal α1,2-linked mannose structures, and eliciting an immunogenic response to this epitope that can cross-react to gp120.
Two approaches have taken to construct homogenous HIV-1 glycopeptides for establishing glycopeptide-based HIV vaccines. Wang, Curr. Opin. Drug Discov. Devel. 9(2):194-206, 2006. These approaches are total chemical synthesis of HIV-1 gp120 glycopeptides carrying either a hybrid-type or a high-mannose-type N-glycan, and chemoenzymatic approach to construct various HIV-1 glycopeptides. See Mandal et al., Angew. Chem. Int. Ed. 43:2557-2561, 2004; and Geng et al., Angew. Chem. Int. Ed. 43:2562-2565, 2004; Singh et al., Bioorg. Med. Chem. Lett. 13:327-330, 2003; Wang et al., ChemBioChem. 6:1068-1074 (2005); Zeng et al., J. Am. Chem. Soc. 127:9692-9693, 2005; Li et al., J. Org. Chem. 70:9990-9996, 2005. However, these synthetic glycopeptides need to be further evaluated for their immunogenicity in animal models.